(1) Field of the Invention
This invention relates to a method for analyzing acidic substances, particularly organic acidic substances, by high speed liquid chromatography.
(2) Description of the Prior Art
Many apparatuses for detecting organic acidic substances by high speed liquid chromatography have been heretofore proposed and put to practical use. These apparatuses have, arranged in series along the path of flow, an eluent supply means comprising an eluent receptacle and a high pressure metering pump; a specimen material supply means comprising a specimen material injector; a column packed with a packing material; and a detector. Many types of detectors are known such as a differential refractometer, a photospectrometer, a thermal detector, a conductivity meter and a solute transport detector. The conventional apparatuses provided with these detectors possess both merits and demerits in analysis properties such as detecting sensibility, universability, selectivity and speed of response.
It is also known to combine a spectrophotometer with a color producing system. For example, a color producing system utilizing dicyclohexyl carbodiimide (DCC), which develops a purplish red color (500-550 nm) under certain conditions, is arranged along the path of flow between the column device and the spectrophotometer-provided detector. This analyzing apparatus exhibits good selectivity and detecting sensibility. However, the color development of DCC involves sequence reactions and, therefore, a plurality of reagent delivery means must be arranged. Thus, the analyzing apparatus is complex and a substantial period of time is necessary for the analysis.
Another example of the combination of a spectrophotometer with a color producing system is described in K. W. Stahl et al: Journal of Chromatographic Science, vol. 10, pages 95-102 (February, 1972). In this color producing system, o-nitrophenol pH indicator is used. Furthermore, an organic solvent is used as the eluent and silica gel is used as the packing material. The o-nitrophenol pH indicator exhibits a visible spectrum such that the areas defined by the convex lines of the transmittance spectrum as measured in alkaline and acidic media overlap with each other to a great degree. Therefore, the o-nitrophenol pH indicator exhibits a poor sensibility when measured on an acidic side. Furthermore, the silica gel used must be treated with sulfuric acid prior to the passage of the eluent through the column. It is difficult or even impossible to completely avoid contamination of the effluent with a trace amount of the remaining sulfuric acid. This unavoidable contamination also reduces the sensibility of the pH indicator and the reproducibility.